Bca protein assay kit

The harvested cells were washed with phosphate buffered saline and lysed with RIPA lysis buffer (Boster, China), which was obtained total cellular protein. The protein concentration was tested by BCA Protein Assay kit (Boster, China). In order to detect the levels of protein expression, the protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a poly-vinylidene fluoride (PVDF) membrane through Bio-Rad II System. The membrane was blocked by 5% skim milk powder at room temperature for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies included caspase-3(Abcam, USA; ab4051), cleaved-caspase-3 (Cell Signaling Technology, USA; #9661), Bax (Abcam, USA; ab182733), Bcl-2 (Abcam, USA; ab692), IL-6R (Boster, China; A01425-1), β-actin (Bioss, China; bs-0061R), and GAPDH (Abcam, USA; ab8245) at 1:500 dilutions. Subsequently, membranes were washed with TBST and incubated with a 1:5000 dilution of HRP-conjugated secondary antibodies (Abcam, USA) for 1 h at room temperature. The protein bands were visualized by ChemiDocTMMP imaging system (Bio-Rad, USA). The GAPDH and β-actin were used as inner loading control, respectively. The gray value was analyzed by Image-ProPlus software.

Proteins were extracted from cells or tissues and added to the RIPA buffer. Cracking on ice for 30_60 minutes. The supernatant was collected by centrifugation at 4°C and 12000 rpm for 15 minutes. BCA protein assay kit (Boster Inc, China) was used for protein quantification. SDS-PAGE gel electrophoresis was performed. Then, the protein was transferred to the nitrocellulose membrane and 5% skimmed milk powder blocked the nonspecific antigen. The primary antibody was added and incubated at 4°C overnight. After washing the membrane, horseradish peroxidase-labeled IgG (secondary antibody) was added at 37 °C for 1h. Densitometric analysis was performed by Image Lab. In the assay, antibodies included rabbit anti-hnRNP E1 (Abcam, UK. ab74793, 1:1000), mouse anti-HPV16 E2 (Abcam, UK. ab17185, 1:1000), mouse anti-HPV16 E6 (Abcam, UK. Ab70, 1:1000), mouse anti-β-actin (Boster Inc, China, 1:300).

Tissues or cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium dexycholate, and 1 mM EDTA, pH8.0) with freshprotease/phosphatase inhibitor cocktail (Boster, Wuhan, China) for 30 min on ice and centrifuged at 16,000 g at 4 °C for 15 min. The protein concentration in supernatant was measured by a BCA protein assay kit (Boster, Wuhan, China). Proteins in the supernatant were separated by SDS-PAGE (Bio-Rad, Richmond, CA, USA) and then, transferred to PVDF membranes (Bio-Rad, Richmond, CA, USA) followed by the blockage in 5% skim milk for 2 h at room temperature. After the membranes were incubated with primary antibodies at 4 °C overnight, HRP-conjugated secondary antibodies were incubated with membrane for 1 h at room temperature. The enhanced chemiluminescence substrate (Boster, Wuhan, China) was applied for color development. The primary antibody against CaMKIIδ was purchased from Abcam (ab105502, Abcam, Cambrige, UK), the antibody against myosin from Millipore (05–716, monoclonal mouse IgG specific for different myosin heavy chain, Millipore Corporation, Billerica, MA, USA), and the antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Santa Cruze Biotechnology (sc-25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

When SK-HEP-1 and MCF-7 cells grew to a density of about 80%, we collected them and added cell lysate buffer (Beyotime, Shanghai, China) and isolated the protein. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). After SDS–PAGE electrophoresis, proteins were transferred to PVDF membrane. The membrane was incubated with the anti-S1PR1 antibody (Abcam, ab233386) for one night and then an anti-rabbit secondary antibody (Sanjian, Tianjin, China, LK2001) for 2 h. Images were processed with Image J software.

After cells or tissues were lysed, we used a BCA Protein Assay Kit (Boster Bio, USA) to determine the protein concentration. Total protein was separated by SDS-PAGE. We used a constant current to transfer the separated protein to a PVDF membrane. 5% skim milk was applied to block non-specific reaction sites for 1-2 h. Dilute the primary antibody at the concentration recommended by the instructions, immerse the membrane in this liquid, and place at 4° C for at least 8 hours. The primary antibodies: anti-GAPDH (Proteintech, Wuhan, China), anti-p-ERK (CST), anti-ERK (CST), anti-NOX4 (Abcam, Cambridge, UK), anti-p-AMPK (Abcam), anti-AMPK (Abcam). Horseradish peroxidase-conjugated secondary antibodies were diluted at the concentrations recommended by the instructions, and the membranes were incubated for 2h at room temperature. Enhanced chemiluminescence reagent was used to visualize the protein bands in an imaging densitometer (GE, USA).

Tris, phosphatidylserine, (R.S)-3-hydroxy-3-methylglutaryl coenzyme A [(R.S)-HMG-CoA], nicotinamide adenine dinucleotide phosphate (NADPH), 1,2-glyceryl dioleate, lecithin, and 3-oleic acid glycerol were purchased from Sigma-Aldrich (St. Louis, MO, USA). [1-¹⁴C] oleoyl-CoA was purchased from New England Nuclear Corporation (Boston, USA). Scintillation solution was purchased from Lipoluma, Lumac Co (Clanton, USA). BCA protein assay kit was from Boster Biological Technology (Wuhan, China). Pravastatin was obtained from Bristol-Myers Squibb (Shanghai, China). Other reagents were obtained from commercial sources.